JP5188523B2 - Bone formation promoter and bone resorption inhibitor - Google Patents

Description

  The present invention relates to a bone formation promotion and bone resorption inhibitor comprising as an active ingredient any one or more of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin. The present invention also relates to a food / beverage product, pharmaceutical or feed for promoting bone formation and suppressing bone resorption, which contains at least one of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin.

  In recent years, various bone diseases such as osteoporosis, fractures, and back pain tend to increase as the elderly population increases. In bone tissue, bone formation and bone resorption are constantly carried out, and the balance between bone formation and bone resorption is maintained when young, but the balance tends to bone resorption due to various causes (aging) Coupling). If this state continues for a long period of time, the bone tissue becomes brittle, and various bone diseases such as osteoporosis, fractures, and back pain occur. If this uncoupling can be prevented, it is considered that various bone diseases such as osteoporosis, fracture, and back pain can be prevented.

Conventionally, as methods for preventing uncoupling and preventing or treating various bone diseases, (1) dietary calcium supplementation, (2) light exercise, (3) sunbathing, (4) drug treatment, and the like have been performed. Yes. For calcium supplementation with diet, calcium salts such as calcium carbonate and calcium phosphate, and natural calcium preparations such as eggshell and fish bone powder are used. However, these are not necessarily materials suitable for oral intake.
Light exercise is good for jogging or taking a walk, but if the body is weak, light exercise is troublesome, and even a bedridden elderly person can hardly exercise. Sunbathing is considered good in terms of supplementing active vitamin D ₃ , but it is not enough. For drug administration, 1α-hydroxyvitamin D ₃ , calcitonin preparation and the like are used, and it is known that they are effective for the treatment of osteoporosis. However, these substances are medicines themselves and cannot be used as food materials.

  On the other hand, the present inventors have continued to search for bone strengthening and bone resorption inhibiting factors present in whey protein in order to obtain a substance having bone strengthening action and bone resorption inhibiting action that can be used as a food material. . As a result, it was found that a protein and peptide mixture obtained by removing whey-derived salts from a water-soluble fraction of whey protein by treatment such as reverse osmosis membrane or electrodialysis has a bone strengthening action (for example, Patent Document 1). reference.). And, it was found that the fraction obtained by subjecting the aqueous solution of the protein and peptide mixture to ethanol treatment, heat treatment, salt treatment, and ultrafiltration membrane treatment has an osteoblast proliferation promoting action and a bone strengthening action (for example, (See Patent Documents 2 and 3.) Moreover, it discovered that the basic protein which exists only in trace amount in milk has an osteoblast proliferation promotion effect, a bone reinforcement effect | action, and a bone resorption suppression effect (for example, refer patent document 4).

The present inventors further advanced the search, tried to separate and purify a component having bone formation promotion and bone resorption inhibitory activity, and identified the substance. Prothrombin, cytochrome P-450, plasminogen, which are known substances, It was confirmed to be transferrin, thrombin and plasmin. And it discovered that these substances could be utilized as a bone formation promotion and bone resorption inhibitor, and came to complete this invention.
Prothrombin is a protein having a molecular weight of about 7.2 kDa and is also called blood coagulation factor II. It is one of vitamin K-dependent blood coagulation factors, and is decomposed into thrombin by factor X in the presence of vitamin K. The generated thrombin decomposes fibrinogen into fibrin, and the blood is coagulated by fibrin cross-linking. Prothrombin is known to be widely distributed in whole body tissues including blood, and it has been reported that it is also present in milk in humans and cows.
The effects of prothrombin and thrombin on bone such as bone strengthening and bone resorption suppression have not been reported so far.

  Cytochrome P-450 (Cytochrome P-450) is a generic name for a group of protoheme proteins that bind to carbon monoxide in a reduced form and show a Soret absorption band near 450 nm. Functionally, various steroid hormones, bile acids, prostanoid synthesis / decomposition reactions, fatty acid ω oxidation, vitamin D activation reactions, oxidative effects of numerous foreign drugs and environmental pollutants taken into the body Involved in detoxification reactions.

  Plasminogen is a precursor of plasmin, exists in animal plasma, and plasminogen activator cleaves the Arg-Val bond in the molecule into active plasmin. It is a single-chain glycoprotein having a molecular weight of about 91,000, and a peptide on the N-terminal side is cleaved by a protease such as plasmin to form a modified plasminogen having a molecular weight of about 82,000.

  Transferrin is a protein with a molecular weight of about 75,000 that binds to transported iron in the blood, and is also called iron-binding globulin. Immature erythrocytes have receptors for iron bound to transferrin, and serum iron must be bound to transferrin in order to be used for hemoglobin synthesis. Regarding transferrin, it has been disclosed that a membrane-bound transferrin-like protein promotes the formation of chondrocytes, but it is the promotion of chondrocyte formation, and the mechanism is completely different from the promotion of bone formation (for example, Patent Documents 5 and 6). reference.).

  There is no literature describing the action of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin on promoting bone formation or inhibiting bone resorption.

Japanese Unexamined Patent Publication No. 4-183371 Japanese Patent Laid-Open No. 5-177715 Japanese Patent Laid-Open No. 5-320066 JP-A-8-15131 JP 2002-20311 A Re-published patent WO01 / 013951

  The present invention provides a bone formation promotion and bone resorption inhibitor that can be used as a food material, can be taken orally for a long period of time, is a safe substance, and has a bone formation promotion and bone resorption suppression action. This is the issue. Moreover, this invention makes it a subject to obtain the food-drinks, pharmaceuticals, or feed for bone formation promotion and bone resorption suppression.

In view of these problems, the present inventors have eagerly and continuously searched for substances that have a bone strengthening action widely contained in food materials, and have a protein having an osteoblast proliferation promoting action and a bone resorption inhibiting action. Attempts were made to isolate and purify components that have bone formation promotion and bone resorption suppression effects, and the substances were identified and confirmed to be known substances prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin. did. And it discovered that these substances can be utilized as an osteoblast proliferation promotion and a bone resorption inhibitor, and came to complete this invention.
That is, the present invention relates to a bone formation promotion and bone resorption inhibitor comprising as an active ingredient any one or more of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin.
The present invention also relates to a food / beverage product, pharmaceutical or feed for promoting bone formation and suppressing bone resorption, which contains at least one of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin.

  The bone formation promoting and bone resorption inhibitor comprising any one or more of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin, and plasmin of the present invention as an osteoporosis, etc. It is useful for the prevention and improvement of various bone diseases. In addition, when this active ingredient is blended in foods, drinks, medicines, feeds, etc., bone formation is promoted, bone resorption is suppressed, and various bone diseases such as osteoporosis are prevented and improved.

  Osteoclasts are cells that originate from hematopoietic stem cells and are present on the surface of cancellous bone and lyse bone. It is believed that osteoclasts dissolve bone matrix (bone resorption), and then osteoblasts synthesize bone matrix, resulting in bone formation and growth (modeling) and metabolism (remodeling). The present invention promotes bone formation by osteoblasts by orally administering at least one of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin, and bone resorption by osteoclasts It has been found that it is remarkably suppressed.

  Expected to balance both bone modeling and remodeling by using the bone formation-promoting and bone resorption-inhibiting effects found in prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin it can. Therefore, this invention provides the bone formation promotion and bone resorption inhibitor, food / beverage products, medicine, or feed which have both a bone formation promotion effect and a bone resorption suppression effect.

  Prothrombin, cytochrome P-450, plasminogen, and transferrin used as active ingredients in the present invention are all present in trace amounts in milk such as cows, buffalo, humans, pigs, sheep, goats, horses, Separation and extraction from milk is possible. As the milk component, a protein fraction separated from raw milk, skim milk or whey, or a cell fraction in milk separated from raw milk can be used. Prothrombin, cytochrome P-450, plasminogen, and transferrin can also be prepared from healthy domestic blood. Prothrombin, cytochrome P-450, plasminogen, and transferrin are all commercially available, and these commercially available products can also be used. Moreover, you may use the recombinant goods produced by genetic engineering.

  Further, prothrombin, cytochrome P-450, plasminogen, and transferrin may be decomposed with a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease. For example, the prothrombin degradation product is a peptide mixture obtained by limited degradation of the above prothrombin with a proteolytic enzyme. Thrombin obtained by decomposing prothrombin with an enzyme such as Factor X may be used. Alternatively, plasmin obtained by decomposing plasminogen with an enzyme such as plasminogen activator may be used. Thrombin and plasmin are also commercially available in the same manner as prothrombin and plasminogen, and these commercially available products can also be used.

For preparation from milk, for example, fresh milk is contacted with an anion exchange resin to adsorb fractions containing prothrombin, cytochrome P-450, plasminogen, and transferrin, and gradually increase the concentration of sodium chloride. After elution, prothrombin, cytochrome P-450, plasminogen, and transferrin can be obtained by gel filtration chromatography.
For preparation from blood, citrate plasma is reacted with barium citrate and the precipitates of prothrombin, cytochrome P-450, plasminogen and transferrin adsorbed on insoluble barium are recovered. Furthermore, the purity can be increased by ion exchange chromatography.

  The bone formation promotion and bone resorption inhibitor of the present invention contains at least one of prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin as an active ingredient. In addition, any one or more of this prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin are used for food and drink such as milk, milk drink, coffee drink, juice, jelly, biscuits, bread, noodles and sausage. It may be blended in or as a medicine such as a tablet or powder. Furthermore, the bone strengthening action can be further enhanced by using a calcium agent with good absorbability such as calcium chloride, calcium carbonate, calcium lactate, eggshell and milk-derived calcium.

  The dose of the bone formation promoting and bone resorption inhibitor of the present invention varies depending on the active ingredient, age, therapeutic effect and disease state, etc. In the case of adults, 1 ng to 100 mg per day should be taken orally in several divided doses. It ’s fine. Thus, various bone diseases such as osteoporosis can be prevented or improved by taking the bone formation promoting and bone resorption inhibitor of the present invention. Prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin were originally derived from plasma and milk, and no acute toxicity was observed in rats. Moreover, these active ingredients can be contained in feed to promote bone formation of livestock, poultry, etc., or to suppress bone resorption.

  EXAMPLES Examples and test examples are shown below, and the present invention will be described in more detail. However, these are merely illustrative, and the present invention is not limited by these.

Bovine plasma prothrombin was prepared according to a known method (Hashimoto, N., T. Morita, and S. Iwanaga, J. Biochem. (Tokyo), vol. 97, p. 1347-1355, 1985). That is, sodium citrate was added to 0.01 M, and bovine plasma was obtained by centrifugation. Barium citrate was slowly added to 10 l of bovine plasma so that the concentration was 0.1 M, and the mixture was gently stirred for 1 hour and then centrifuged at 4,000 rpm for 10 minutes. The obtained precipitate was washed with a 0.1 M sodium chloride solution containing 0.01 M barium chloride, 1 mM benzamidine hydrochloride and 0.02% sodium azide, and centrifuged. This washing operation was repeated twice. The barium precipitate was suspended in 1 l of 40% saturated ammonium sulfate, diisoprolyl phosphorofluoridate (DFP) was added to 1 mM, and the mixture was stirred overnight. Centrifugation was performed at 5,000 rpm for 30 minutes to obtain a supernatant. Saturated ammonium sulfate was added to this supernatant to 67% saturation, and the precipitate was recovered by centrifugation at 5,000 rpm for 30 minutes. This precipitate was dissolved in 100 to 150 ml of 0.05 M sodium phosphate buffer (pH 6.0) containing 0.2 M sodium chloride, 1 mM DFP and 1 mM benzamidine hydrochloride, and dialyzed overnight against 20 l of the same buffer. Insoluble components were removed by centrifugation, and then subjected to DEAE-Sephadex A50 chromatography. The sample was applied to a DEAE-Sephadex A-50 column (200 x 100 mm) equilibrated with 0.05 M sodium phosphate buffer (pH 6.0) containing 0.2 M sodium chloride and 1 mM benzamidine hydrochloride, and the sodium chloride concentration was 600 mM. And the adsorbed protein was eluted. Prothrombin was eluted when the sodium chloride concentration reached about 300 mM. This was desalted by dialysis and then applied to a Blue Sepharose CL-6B column (80 × 200 mm) equilibrated with 50 mM Tris-HCl buffer (pH 6.3) containing 0.1 M sodium chloride and 1 mM benzamidine hydrochloride. After washing with the equilibration buffer, the adsorbed protein was eluted with a buffer with a sodium chloride concentration of 4 M. This was desalted by dialysis and freeze-dried to obtain 1.0 g of prothrombin, which is an active ingredient of the present invention. 1.0 g of this prothrombin was mixed with 9.0 g of lactose and molded into granules to obtain the bone formation promotion and bone resorption inhibitor of the present invention.
Note that cytochrome P-450, plasminogen, and transferrin can also be prepared from bovine plasma by the same method as for prothrombin.

  A fraction containing prothrombin was prepared from milk. A column (5 cm in diameter x 30 cm in height) packed with 400 g of the cation exchange resin SP Sepharose HP (Amersham Biosciences) was thoroughly washed with deionized water, and then 40 l of non-sterilized skim milk ( pH 6.7) was passed at a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water, and the protein fraction adsorbed on the resin was eluted with 0.02 M carbonate buffer (pH 7.0) containing 1.5 M sodium chloride. This eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and then lyophilized to obtain 21 g of protein fraction powder, which was used as the bone formation promotion and bone resorption inhibitor of the present invention. This protein fraction powder contained 0.01% by weight of prothrombin.

[Test Example 1]
Prothrombin, cytochrome P-450, plasminogen, transferrin, thrombin and plasmin were examined for osteoblast proliferation. Each active ingredient used in the test was commercially available. The origin of each active ingredient subjected to the test and the final concentration of each active ingredient are shown in Table 1.
Mouse osteoblasts MC3T3-E1 cells are seeded in 96-well plates with α-MEM medium (manufactured by Flow Laboratories) containing 10% fetal bovine serum at a cell count of 2 × 10 ⁴ / ml and 5% CO _{2 is} present Then, the cells were cultured at 37 ° C. for 24 hours to obtain cultured cells for testing. Then, the medium is replaced with α-MEM medium not containing fetal bovine serum, and prothrombin, cytochrome P-450, plasminogen, transferrin, plasmin, and thrombin are added to the medium so that the final concentrations shown in Table 1 are obtained. And then cultured at 37 ° C. for 18 hours. Cell proliferation activity was examined with a cell profilation assay kit (Amersham Biosciences) using bromodeoxyuridine (BrdU). As a control, an active ingredient-free additive was used, and the cell proliferation activity (%) of the additive-free additive was defined as 100%. The results are shown in Table 1.

  Prothrombin, cytochrome P-450, plasminogen, transferrin, plasmin, and thrombin all have an increased cell proliferative activity compared to those without any active ingredient, and are found to have osteoblast proliferative activity. It was.

[Test Example 2]
Bone resorption inhibitory effects of prothrombin, cytochrome P-450, plasminogen, transferrin, plasmin and thrombin were examined. Each active ingredient used in the test was commercially available. The origin of each active ingredient subjected to the test and the final concentration of each active ingredient are shown in Table 2.
After removing the long bones of 10-20 days old ICR mice, removing soft tissues, mechanically chopping bones in α-MEM (Flow Laboratories) solution containing 5% fetal calf serum And whole bone marrow cells containing osteoclasts were obtained. The whole bone marrow cells containing osteoclasts were seeded on ivory pieces so as to be about 2 × 10 ⁶ cells, and spotted with an α-MEM solution containing 5% fetal calf serum. Two hours later, prothrombin, cytochrome P-450, plasminogen, transferrin, plasmin and thrombin were added to an α-MEM solution containing 5% fetal bovine serum so that the final concentrations shown in Table 2 were obtained. After culturing for one day, the bone resorption inhibitory activity of existing osteoclasts was examined.
To evaluate the bone resorption inhibitory action, after culturing, the cells on the ivory pieces were peeled off and stained with hematoxylin, and image analysis was performed with PIASLA-555 to measure the number of bone resorption pits. (Kenji Seno et al., Animal Culture Cell Manual by Research Theme, pp.199-200, 1993). That is, the small number of pits means that the activity of osteoclasts was reduced and the bone resorption activity was suppressed. In addition, substances that have been shown to inhibit bone resorption by pit assay have also been shown to inhibit bone resorption in animal experiments (Toba et al., Bone, vol.27, p.403-408, 2000). The pit assay is a suitable experimental system for examining the bone resorption inhibitory effect. As a control, an active ingredient-free additive was used, and the bone-resorbing activity (%) of each additive-free additive was defined as 100%. The results are shown in Table 2.

  Any of the active ingredients of the present invention to which prothrombin, cytochrome P-450, plasminogen, transferrin, plasmin and thrombin are added has suppressed bone resorption activity compared to those without any active ingredient, and is remarkable. It was found to have a bone resorption inhibitory action.

  After mixing the raw materials with the formulation shown in Table 3, the container was filled and sterilized by heating to produce a juice drink for promoting bone formation and suppressing bone resorption.

  After mixing the raw materials with the formulation shown in Table 4, the tablet was pressed by a tableting machine to produce a tablet-type bone formation promoting and bone resorption inhibitor.

  The ingredients shown in Table 5 were mixed using commercially available plasminogen, the dough was created and molded, and then baked to produce bone formation promotion and bone resorption suppression biscuits.

  The raw materials were mixed using commercially available plasmin with the formulation shown in Table 6 to produce bone formation promotion and bone resorption suppression biscuits.

  After mixing the raw materials with the formulation shown in Table 7, the container was filled and sterilized by heating to produce a jelly for promoting bone formation and suppressing bone resorption.

  The raw materials were mixed using commercially available prothrombin with the formulation shown in Table 8, and then emulsified at 85 ° C. to produce a process cheese for promoting bone formation and suppressing bone resorption.

  The raw materials were mixed using commercially available thrombin with the formulation shown in Table 9 to produce a process cheese for promoting bone formation and suppressing bone resorption.

  12% by weight reduced skim milk was sterilized by heating at 90 ° C for 20 minutes, then inoculated with Lactobacillus acidophilus and Streptococcus thermophilus to obtain two types of starter cultures, etc. The amount was mixed. And after mixing a raw material with the mixing | blending shown in Table 10, it was made to ferment and the yoghurt for bone formation promotion and bone resorption suppression was manufactured.

  The raw materials were mixed using the prothrombin obtained in Example 1 with the formulation shown in Table 11 to produce infant formula for promoting bone formation and suppressing bone resorption.

  The raw materials were mixed using commercially available thrombin with the formulation shown in Table 12 to produce infant formula for promoting bone formation and suppressing bone resorption.

  Raw materials were mixed using commercially available plasminogen with the formulation shown in Table 13 to produce a dog breeding feed for promoting bone formation and suppressing bone resorption (dog food).

  The raw materials were mixed using commercially available plasmin with the formulation shown in Table 14 to produce a dog breeding feed for promoting bone formation and suppressing bone resorption (dog food).

Claims (3)

  A bone formation promoting and bone resorption inhibitor comprising plasminogen and / or plasmin as an active ingredient.   The bone formation promotion and bone resorption inhibitor according to claim 1, wherein 1 ng to 100 mg is administered per day. A pharmaceutical composition for promoting bone formation and inhibiting bone resorption, comprising the bone formation promoting and bone resorption inhibitor according to claim 1 or 2.